Molecular cloning and characterization of cold-responsive gene <Emphasis Type="Italic">Cbrci35</Emphasis> from <Emphasis Type="Italic">Capsella bursa-pastoris</Emphasis>

A new rare cold-inducible (RCI) gene designated Cbrci35 was cloned from Capsella bursa-pastoris, an edible wild herb, using the rapid amplification of cDNA ends (RACE) method. The full-length cDNA of Cbrci35 (Database Accession No.: AY566573) was 1300 bp and contained a 978 bp ORF encoding a precursor of 326 amino acid residues with a 23 amino acids signal peptide. The predicted Cbrci35 protein contained a peroxidase active site and proximal heme-ligand signatures, an RGD cell attachment sequence motif and two leucine zipper pattern motifs. Bioinformatics analysis revealed that Cbrci35 has a high level of similarity with RCI genes from Arabidopsis thaliana and peroxidases genes from other plants. RT-PCR analysis revealed that Cbrci35 expressed only in root. A cold acclimation assay showed that Cbrci35 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. But expression was not induced exposed to dehydration, salt stress or abscisic acid, indicating that it might be subjected specifically to cold regulation. These results indicate that Cbrci35 is an analogue of RCI genes and may participate in cold-response or increasing the freezing tolerance of plants.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.     

The <Emphasis Type="Italic">Arxula adeninivorans</Emphasis><Emphasis Type="Italic">ATAL</Emphasis> gene encoding transaldolase-gene characterization and biotechnological exploitation

The yeast Arxula adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins. In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for the expression of a human serum albumin (HSA) reporter gene.     

Harvest-inducibility of the promoter of alfalfa<Emphasis Type="Italic"> S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine:<Emphasis Type="Italic"> trans</Emphasis>-caffeoyl-CoA3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase gene

A major limitation on the expression of some foreign proteins in transgenic plants is the toxic effect of such proteins on the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems. A cDNA clone was obtained from subtractive hybridization of non-harvested and harvested alfalfa leaf tissue, named hi12. The hi12 cDNA was identified as part of the S-adenosyl-l-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa, a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was strongly induced by harvesting and wounding but not by heat shock. The promoter of the hi12 gene, isolated by genomic walking, contained several stress response cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the GUS gene driven by the promoter showed GUS expression following harvesting, demonstrating the activity of these regulatory regions in other plant species.     

Molecular cloning,characterization and expression of a chalcone reductase gene from <Emphasis Type="Italic">Astragalus membranaceus</Emphasis> Bge. var. <Emphasis Type="Italic">mongholicus</Emphasis> (Bge.) Hsiao

A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein of 35 kDa, a 67 bp 5′ non-coding region and a 172 bp 3′-untranslated region. The putative AmCHR protein showed striking similarity to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38% extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa, which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-β-d-glucoside branch pathway in A. mongholicus.     

Isolation and characterization of a novel glycogen synthase kinase-3 gene, <Emphasis Type="Italic">GmGSK</Emphasis>, in <Emphasis Type="Italic">Glycine max</Emphasis> L. that enhances abiotic stress tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A novel glycogen synthase kinase-3 gene, GmGSK, was isolated from Glycine max. It is 1,596 bp in length with one ORF of 410 amino acids. Southern blot analysis revealed that it has at least two copies in the G. max genome. GmGSK, when transiently expressed in Nicotiana tabacum leaves, was localized in both cell membrane and cytoplasm. Northern blot analysis indicated that GmGSK is expressed in all tissues, with highest expression in the root. GmGSK can be induced by various abiotic stresses. When transformed with GmGSK, Saccharomyces cerevisiae exhibited enhanced resistance to salt and drought stress.